Isolation of cellular material under microscopic visualization

ABSTRACT

A method of microdissection which involves: forming an image field of cells of the tissue sample utilizing a microscope, identifying at least one zone of cells of interest from the image field of cells which at least one zone of cells of interest includes different types of cells than adjacent zones of cells, and extracting the at least one zone of cells of interest from the tissue sample. The extraction is achieved by contacting the tissue sample with a transfer surface that can be selectively activated so that regions thereof adhere to the zone of cells of interest to be extracted. The transfer surface includes an activatable adhesive layer which provides chemical or electrostatic adherence to the selected regions of the tissue sample. After the transfer surface is activated the transfer surface and tissue sample are separated. During separation the zone of cells of interest remains adhered to the transfer surface and is thus separated from the tissue sample.

RELATED APPLICATION

[0001] The present application is a continuation-in-part of U.S. patentapplication Ser. No. 08/203,780, filed Mar. 1, 1994.

TECHNICAL FIELD

[0002] The present invention relates to methods and devices for theanalysis of cellular samples. More particularly, the present inventionrelates to methods and devices for the microdissection and analysis ofcellular samples which may be used in combination with a number ofdifferent technologies that allow for analysis of enzymes, MRNA and DNAfrom pure populations or subpopulations of particular cell types.

BACKGROUND ART

[0003] Many diseases are now understood at the molecular and geneticlevel. Analysis of such molecules is important for disease diagnosis andprognosis. Previous methods for direct extraction of cellular tissuematerial from a tissue sample are limited because the extractionreflects only the average content of disease associated markers. Inreality, tissues are very heterogeneous, and the most diagnosticportions of the tissue may be confined to a few hundred cells or less ina lesion.

[0004] Normal tissue samples contain a variety of cell types surroundingand adjacent to the pre-invasive and invasive tumor cells. A region ofthe tumor tissue subject to biopsy and diagnosis as small as 1.0 mm cancontain normal epithelium, pre-invasive stages of carcinoma, in-situcarcinoma, invasive carcinoma, and inflammatory areas. Consequently,routine scraping and cutting methods will gather all of these types ofcells, and hence, loss of an allele will be masked by presence of anormal copy of the allele in the contaminating nonmalignant cells.Existing methods for cutting away or masking a portion of tissue do nothave the needed resolution. Hence the analysis of genetic results bythose previous are always plagued by contaminating alleles from normalcells, undesired cells or vascular cells.

[0005] The molecular study of human tumors is currently limited by thetechniques and model systems available for their characterization.Studies to quantitatively or qualitatively asses proteins or nucleicacid expression in human tumor cells are compromised by the diversecell. populations present in bulk tumor specimens. Histologic fields ofinvasive tumor typically show a number of cell types including tumorcells, stromal cells, endothelial cells, normal epithelial cells andinflammatory cells. Since the tumor cells are often a relatively smallpercentage of the total cell population it is difficult to interpret thesignificance of net protein or nucleic acid alterations in thesespecimens.

[0006] The processes of tumor invasion and metastasis depend uponincreased proteolytic activity of invading tumor cells. Matrixmetalloproteinases, cathepsins B, D, and L, and plasminogen activatorhave been implicated in the metastatic cascade. Cathepsin D has beensuggested to be an independent marker of prognosis in breast cancer.Several lines of correlation evidence support the concept that proteasesare important in tumor invasion including: increased protease activityand/or altered subcellular distribution of proteases in highlymetastatic tumor cell lines, increased protease expression in invasivehuman tumors as determined by both immunohistochemistry and assays oftumor tissue homogenates, and increased MRNA levels in human tumors. Allof these techniques have generated important information regardingprotease expression in human tumors, however, they have not provideddefinitive evidence that proteases are up-regulated in specific regionswhere tumor invasion is occurring.

[0007] Studies of human tumor cells in culture do not account for thecomplex interactions of the tumor cells with host cells andextracellular matrix, and how they may regulate tumor cell proteaseproductivity or activation. Immunohistochemical staining allows one toexamine enzyme distribution in regions of tumor invasion, however,results vary with tissue fixation and antibody-antigen affinity, andprovide only a semi-quantitative assessment of protein levels.Furthermore, quantitative interpretation of staining results iscomplicated by the variability of staining patterns within tissuesections, subjective evaluation of staining intensity, and thedifficulty in interpreting the, significance of stromal staining. Inaddition, many antibodies utilized in the study of proteases do notdifferentiate pro-enzyme from active enzyme species. Assays of enzyme orMRNA levels from homogenates of human tumors does not account for eitherthe mixed population of cells within the specimens, or the concomitantpathophysiologic processes which may be occur in the tissue.

[0008] Human tumors accumulate genetic abnormalities as they developfrom a single transformed cell to invasive and metastatic carcinoma.Identification and characterization of the genes which are mutated, lostor abnormally regulated can provide important insights for cancerdiagnosis, prognosis, and therapy. Furthermore, identification of suchgenetic lesions may facilitate early diagnosis by definitiveidentification of premalignant lesions so they can be treated beforethey progress to invasive cancer.

[0009] A general dictum of cancer progression states that cells can betransformed after acquiring two separate alterations in the tumorsuppressor gene. Subsequent tumors progress stepwise from dysplasticlesions to in-situ, to invasive and metastatic neoplasms. In-situcarcinomas are frequently observed arising in association with aspectrum of epithelial hyperplasias and larger invasive tumors are oftenassociated with regions of carcinoma in-situ at the tumor periphery.

[0010] Pathologists have historically interpreted a side-by-sideassociation of atypical hyperplasia, in-situ carcinoma, and invasivetumors as evidence of a cause and effect relationship among theentities. However, little direct evidence existed previously whichsupports this model.

[0011] Prior methods of study have not allowed investigators tospecifically examine genetic alterations in pre-invasive lesions. Thepresent invention provides a novel improved means to specificallyexamine genetic alterations in pre-invasive lesions of common epithelialtumors such as breast and prostate carcinoma. In particular the presentinvention permits the microsampling of five or less cells with RNA andDNA extraction of the sampled cells. This method has been demonstratedto be extremely sensitive and to surpass previous and currenttechnologies by more than two orders of magnitude. It has allowed thesensitive detection of loss of heterozygosity in early pre-invasivelesions being a gateway to the discovery of a new genetic loci onchromosome 11 for breast cancer and a new genetic loci on chromosome 8for prostate carcinoma.

DISCLOSURE OF THE INVENTION

[0012] It is according one object of the present invention to provide amethod of identifying specific cells in cellular tissue sample.

[0013] Another object of the present invention is to provide a method ofdirect extraction of specific cells from a cellular tissue sample.

[0014] It is a further object of the present invention to provide anautomated method of identifying specific cells in cellular tissuesample.

[0015] A further object of the present invention is to provide anautomated method of direct extraction of specific cells from a cellulartissue sample.

[0016] A still further object of the present invention is to provide amethod of obtaining pure cell populations from a cellular tissuesamples.

[0017] According to these and further objects of the present inventionwhich will become apparent as the description thereof proceeds, thepresent invention provides for a method of direct extraction of cellularmaterial from a tissue sample which involves:

[0018] providing a tissue sample;

[0019] contacting the tissue sample with a transfer surface which can beactivated to provide selective regions thereof with adhesivecharacteristics;

[0020] identifying at least one portion of the tissue sample which is tobe extracted;

[0021] activating a region of the transfer surface which is in contactwith the at least one portion of the tissue sample so that the activatedregion of the transfer surface adheres to the at least one portion ofthe tissue sample; and

[0022] separating the transfer surface from the tissue sample whilemaintaining adhesion between the activated region of the transfersurface and the at least one portion of the tissue sample so that the atleast one portion of the tissue sample is extracted from a remainingportion of the tissue sample.

BRIEF DESCRIPTION OF DRAWINGS

[0023] The present invention will be described with reference to theattached drawings which are given by way of non-limiting examples only,in which:

[0024]FIG. 1 is a functional system diagram which shows how a tissuesample is microscopically imaged, displayed on a display monitor, andhow a region of the imaged sample is selected and identified forsubsequent microdissection and analysis.

[0025]FIGS. 2a-2 c are a series of functional system diagrams which showhow a zone of tissue sample is extracted from the slide-mounted tissuesample according to one embodiment of the present invention.

[0026]FIG. 3 is a schematic illustration of an alternative device forextracting sample zones from the slide-mounted tissue sample.

[0027]FIGS. 4a and 4 b are schematic diagrams of a manual extractiontool manipulator which can be used together with the extraction deviceof FIG. 3 according to the present invention.

[0028]FIG. 5 is a functional system diagram which shows how a zone ofsample tissue can be directed to an appropriate analysis protocol.

[0029]FIGS. 6a and 6 b show the expression of MMP-2 in ten invasivecolon carcinoma cases (FIG. 6a) and in five cases of invasive breastcarcinoma (FIG. 6b) as compared to normal colonic mucosa from the samepatients.

[0030]FIG. 7 shows SSCP analysis of MMP-2 activation site.

[0031]FIGS. 8a-8 d are schematic illustrations of the sequential stepsof an adhesive transfer method according to one embodiment of thepresent invention.

[0032]FIG. 9 shows the results of a sequencing gel electrophoresis ofPCR amplified DNA from human tissue microdissected by the methoddepicted in FIGS. 8a-8 b.

BEST MODE FOR CARRYING OUT THE INVENTION

[0033] The present invention is directed to a method of analyzingcellular material on a molecular or genetic level which involves:visualizing a field of cells in a tissue sample under a microscope,contacting an identified area with a surface which simultaneouslydissolves, extracts and/or retains a cellular material of interest, andtransferring the cellular material of interest to a suitable analysissystem. The present invention is particularly applicable to the analysisof local tissue enzymes, antigens, DNA, RNA, and the like.

[0034] According to a preferred embodiment, the present invention isdirected to adhesive transfer methods which involve microscopicvisualization and transfer of cellular material to a procurement ortransfer surface.

[0035] The present invention is also directed to a fully automatedsystem whereby a tissue can be visualized on a screen, so that a precisefield of cells of interest can be identified by a variety of labels,circumscribed, and then be automatically extracted and analyzed.

[0036]FIG. 1 is a functional system diagram which shows how a tissuesample is microscopically imaged, displayed on a display monitor, andhow a region of the imaged sample is selected and identified forsubsequent microdissection and analysis. As depicted in FIG. 1, a tissuesample 1 is provided on a glass slide 2 for microscopic examination andimaging. The sample tissue 1 can be fixed on the glass slide 2 accordingto any conventional method, including attachment to the glass slide 2with an agarose gel, fixing the tissue sample in paraffin, etc.

[0037] The glass slide 2 having the sample tissue 1 mounted thereon isplaced on the stage of a microscope. The microscope, generally indicatedby reference numeral 3 receives an image of the tissue sample 1. A videocamera (not shown) is connected to the microscope 3. The video camerareceives the image of the sample tissue 1 from the microscope 3 anddisplays the image of the tissue sample on a display monitor 4.

[0038] The image of the sample tissue 1 is limited to the “field” of themicroscope 3 for any given image. As indicated in FIG. 1, the field ofthe sample tissue image may include several zones, “A”, “B”, “C”, and“D” of different types of cells which can be optically distinguished byutilizing a suitable dye(s) to stain the tissue sample. For exemplarypurposes, FIGS. 1 and 2a-2 c assume that zone “B” is the zone ofcellular material of interest. The image on the display monitor 4 isused by the operator to select and identify one or more zones of thetissue sample 1 which are of interest. According to one embodiment ofthe present invention, after the zone(s) of interest are selected andidentified, the operator manually manipulates a device to extract theidentified zone(s) from the glass slide 2. The extracted zone(s) ofsample material may either include an analysis sample. Otherwise, theidentified and extracted zone(s) can include zones which are todiscarded and the remaining zone(s) which are retained on the glassslide 2, can be later analyzed.

[0039] In addition to manual operation which is discussed in more detailbelow, it is possible, according to another embodiment of the presentinvention, to utilize the image on the display monitor 4 to select andidentify a sample zone(s) whose relative position is determinedutilizing a computer which receives a digitized signal of the image fromthe video camera (or microscope), and which receives a referenceposition of the stage of the microscope 3 upon which the sample is held.Such positioning detection and recognition systems are conventional inthe art and can be readily applied to automate the sample preparationmethod of the present invention. In this automated embodiment of theinvention, the computer which performs the positioning detection andrecognizing can also be used to control the movement of the devicesdiscussed below that are used to extract tissue zones, thus automatingthe sample removal. In addition, the image of the sample can beelectronically scanned to automatically identify zones having apredetermined or relevant degree of staining, using known techniques anddevices. Thus, in a preferred embodiment, a computer could be used toselect and identify zones of interest and the relative position of suchzones, for manipulating a device to remove such zones in an automatedmanner.

[0040]FIGS. 2a-2 c are a series of functional system diagrams which showhow a zone of tissue sample 1 is extracted from the slide-mounted tissuesample 1 according to one embodiment of the present invention. It is tobe understood that the steps depicted in FIGS. 2a-2 c could be eitherpreformed manually by an operator or by a computer utilizingconventional positioning and control methods, e.g. computer controlledrobotics.

[0041] The embodiment of the invention depicted in FIGS. 2a-2 c utilizea contact probe 5 which has an adhesive/extraction reagent 6 on the tipthereof. A suitable adhesive/extraction reagent can include a mixture ofpiccolyte and xylene. In FIG. 2a the contact probe 5 is positionedeither manually or by computer control so as to be above and alignedwith the sample zone (“B”) to be extracted. As can be readily understoodfrom FIG. 2a, the surface area of the contact probe tip (andadhesive/extraction reagent) needs to be about equal to, and no greaterthan, the surface area of the zone to be extracted. otherwise, excessiveremoval of adjacent tissue zones will occur.

[0042] Once the tip of the contact probe 5 is aligned with the samplezone (“B”) to be extracted, the contact probe 5 is lowered so that theadhesive/extraction reagent 6 on the tip thereof contacts the samplezone (FIG. 2b).

[0043] The adhesive/extraction reagent 6 is selected to readily adhereto the sample zone. Once the adhesive/extraction reagent 6 on the tip ofthe contact probe 5 contacts the sample zone (FIG. 2b) and the samplezone becomes adhered thereto, the contact probe 5 can be retracted fromthe contact position (illustrated in FIG. 2b) and moved as shown in FIG.2c. Since the relative adhesive force of the adhesive/extraction reagentis greater than the adhesive force used to mount the sample on the glassslide, the contact probe 5 pulls the sample zone “B” from the glassslide when withdrawn or retracted. According to one embodiment of thepresent invention, a glass pipette was used as the contact probe 5. Inthis embodiment, the tip of the glass pipette was coated with a solutionof piccolyte (568 g/l) and xylene (437.5 g/l) by dipping the tip of theglass pipette in the piccolyte/xylene solution.

[0044] In addition to removing the sample zone from the glass slide 2,the contact probe 5 can be used to transfer the extracted sample zone toan analysis container 7 as indicated in FIG. 2c or to any otherlocation, such as a waste container, a culture media, etc. In apreferred embodiment, the contract probe 5 is used to transfer theextracted sample zone to the sample receiving stage of an automatedclinical analyzer which is designed to preform a desired analysis of thesample zone. It thus, can be understand that the present invention canprovide a fully automated method and system for identifying sample zoneson a slide-mounted sample, removing sample zones of interest from theslide-mounted sample, and transporting the extracted sample zones to anautomated analyzer which can perform automated analysis of the extractedsample zones.

[0045] In FIG. 2c the extracted sample zone is depicted as beingdispensed in a container 7 which, for example, can be a test tube orsimilar container in which analysis on the extracted sample zone can beinitiated or performed. As depicted in FIG. 2c, a reagent solution 8which removes all or a desired component of the extracted sample zonefrom the contact probe tip can be placed in the container 7 before theextracted sample zone is deposited therein. For example, in the case ofDNA analysis, a solution of Tris (50 mM, pH8.5), EDTA (1 mM), Tween 20(0.5%), and proteinase K (0.2 mg/mL) can be used to remove the extractedsample zone from the tip of the contact probe 5 and dissolve the tissuematerial for analysis purposes.

[0046] In addition to the contact probe depicted in FIGS. 2a-2 c, ahollow suction probe could also be used to extract sample zones from theslide-mounted tissue sample 1. Such a suction probe could be providedwith sharp annular tip by which sample zones could be punched out andextracted by suction forces.

[0047]FIG. 3 is a schematic illustration of an alternative device forextracting sample zones from the slide-mounted tissue sample 1. Theextraction device 9 shown in FIG. 3 includes a cutting blade 10 and agrasping arm 11. The grasping arm 11 can be moved in an opposed mannerwith respect to the cutting blade 10. The grasping arm 11 is shown inits open position in FIG. 3. The grasping arm 11 is movable between theillustrated open position to a closed position in which the tip of thegrasping arm 11 contacts the cutting blade 10. The movement of thegrasping arm 11 can be controlled by a cable and pulley system in whichgrasping arm 11 is caused to pivot at its base by applying tension to acable which passes through a pulley located at the base of the graspingarm 11. The tension on the cable can be applied by actuating a lever ordepressing a button 12 on the device which applied tension to the cablein a known manner. Such actuating mechanical structures are known in theart of gripping devices.

[0048] In operating the device of FIG. 3, the cutting blade 10, which isat an obtuse with respect to the central axis of the device can cut outand scoop up a portion of a tissue sample by placing the cutting blade10 on one edge of a portion of the tissue sample to be extracted andthen moving the grasping arm 11 into the closed position. As thegrasping arm 11 comes into contact with the tissue sample, it draws thecutting blade 10 into the sample and presses a portion of the sampletoward the cutting blade 10 thereby causing a portion of the samplecontacted between the cutting blade 10 and the grasping arm 11 to be cutout and scooped up from the sample.

[0049] In a further, alternative embodiment of the device of FIG. 3, themovement of the grasping arm 11 can be effected by a toothed gearinstead of a pulley and a cooperating toothed rod in place of a cable.Such mechanical structures are known in the art of gripping devices.

[0050]FIGS. 4a and 4 b are schematic diagrams of a manual extractiontool manipulator which can be used together with the extraction deviceof FIG. 3 according to the present invention. In FIG. 4a the extractiontool manipulator is depicted as having a base 13 equipped with aclamping means 14 for removable attaching the device to a brace orsupport portion of the stage of a microscope (see FIG. 4b). The clampingmechanism includes a clamping plate 15 that is secured to a threadedshaft 16 which passes through a threaded bore 17 in a lower portion ofthe base 13. A tightening knob 18 is provided on the end of the threadedshaft 16. Turning the tightening knob 18 causes the clamping plate 15 tomove with respect to an upper portion 19 of the base 13. Thus, theextraction tool manipulator can be clamped to a portion of the stage ofa microscope 20 as depicted in FIG. 4b by positioning a brace or supportportion 21 of the stage of the microscope 20 between the clamping plate15 and the upper portion 19 of the base 13 and turning knob 18 totighten the clamping plate 15 against the brace or support portion 21 ofthe stage of the microscope 20.

[0051] The extraction tool manipulator includes a tool holder 22 havinga through-bore 23 therein for receiving the shaft of an extraction tool24. Ideally, the tool holder 22 should allow for damped fore and aftmovement of the extraction tool. Therefore, according to a preferredembodiment, the through-bore 23 of the tool holder 22 contains a bushingwhich can be adjustable tightened against the tool shaft by tool lockingscrew 24.

[0052] The tool holder 22 is supported by support shaft 25 which isconnected at opposite ends by 360° damped swivels 26 and 27 to the toolholder 22 and the base 13. The length of the support shaft 25 betweenthe 360° damped swivels 26 and 27 is adjustable. The adjustment of theindependent 360° damped swivels 26 and 27 together with the adjustablelength of the support shaft 25 and the position of the tool shaft withinthrough-bore 23, allows a high degree of movement of the extraction toolwith respect to a slide-mounted sample positioned on the stage of themicroscope. Therefore, an operator can manipulate an extraction toolheld by the extraction tool manipulator and remove selected tissue zonesfrom a slide-mounted tissue sample with a high degree of precision.

[0053]FIG. 5 is a functional system diagram which shows how a zone ofsample tissue can be directed to an appropriate analysis protocol. Asdepicted in FIG. 5 a microextraction of a zone of tissue sample can betaken from a slide-mounted tissue sample 1 as discussed above andtransferred to a sample preparation stage 28 in which the cells ofinterest can be extracted and collected for analysis. Excised cells mayalso be solubilized at this stage. If the cells of interest contain DNAor RNA the extracted sample is subjected to polymerase chain reaction(PCR) amplification and hybridization, strand conformationalpolymorphism, and southern and northern blotting as desired. If thecells of interest contain proteins, the extracted sample can besubjected to enzyme zymography, an immunoassay, or a biochemical assay.

[0054] Selective extraction or microdissection of frozen tissue sectionsaccording to the present invention allows for recovery and analysis ofboth active enzymes and MRNA. Additionally, the DNA recovered from thesesections is in the native condition and can be used for studies such asDNA fingerprinting. Microdissection of paraffin embedded tissuesaccording to the present invention allows for PCR amplification of DNAfrom pure cell populations representing less than one high poweredfield, or a single layer of epithelial cells lining cystic spaces.

[0055] For general preparation of samples for frozen sectionmicrodissection according to the present invention microdissectionslides can be prepared by placing 1% agarose on a standard histologyslide and cover slipping. After a short period of time, e.g., 5 minutesthe cover slip is removed leaving a thin gel on the slide. A smallfrozen tissue section, e.g. 25 micron thick, is placed on the agarosegel and briefly stained with eosin. The tissue may also be treated withagents to denature RNase depending on the subsequent extraction method.Under direct microscopic visualization the specific cell population orsub-population of interest is procured from the tissue section utilizingthe techniques discussed above.

[0056] For enzyme analysis the procured tissue specimen can be placed inan appropriate buffer depending on the enzyme of interest. The enzymelevels can be measured by several methods including zymography and theuse of specific fluorometric substrates. The precise levels of enzymeexpression in a specific cell population can be determined.

[0057] For messenger RNA analysis the tissue specimen can be placed onagarose and treated with agents to denature RNase if necessary. Theprocured tissue specimen is immediately frozen in liquid nitrogen. Thetissue can be used immediately or stored at −70° C. for several months.The MRNA can be extracted using an oligo dT column (Micro-Fast trackMRNA Isolation Kit, Invetrogen Co.). The recovered MRNA of the pure cellpopulations can be amplified and investigated using PCR technology.

[0058] For DNA analysis the tissue specimen can be placed in a singlestep extraction buffer solution of 50 mM Tris, pH 8.5; 1 mM EDTA, 0.5%Tween 20, and 0.2 mg/ml proteinase K, incubated for four hours at 37°C., followed by ten minutes incubation at 95° C. The recovered DNA canbe amplified and analyzed by PCR technology. If native DNA is requiredfor DNA fingerprinting, the proteinase K can be added after DNase.

[0059] For paraffin section microdissection routine formalin fixed,paraffin embedded tissue sections are microdissected afterdeparaffinization and brief staining with eosin. Tissue sections arevisualized by direct microscopy and cell populations or subpopulationsof interest are procured using a modified glass pipette with theadhesive coated tip discussed above. Tissue specimens as small as onecell can be procured with this method. The specificity of dissectionrepresents a significant improvement over currently known techniques.

[0060] For DNA analysis of paraffin embedded tissue, the glass pipettewith the dissected tissue specimen is placed in a single step extractionbuffer solution of 50 mM Tris, pH 8.5; 1 mM EDTA, 0.5% Tween 20, and 0.2mg/ml proteinase K which removes the tissue from the pipette tip.Depending on the size of the sample it is incubated from two totwenty-four hours at 37° C., followed by a ten minute incubation at 95°C. The glass pipette tip can then be sterilized and reused.

[0061] Features and characteristics of the present invention will beillustrated by the following examples to which the present invention isnot to be considered limited. In the examples and throughout percentagesare by weight unless otherwise indicated.

[0062] The following examples were performed in an attempt to establishif the present invention could be used to more specifically studyprotease distribution during human tumor invasion. Levels of MMP-2 andcathepsin B in fields of invasive breast and colon carcinoma weremeasured to assess if the enzymes in these regions were quantitativelyincreased as compared to matched numbers of normal cells from the samepatient.

[0063] In the following examples, normal and tumor samples of colon andbreast tissue frog surgical resections were maintained in a frozencondition (−70° C.) until analysis. Tissue section of invasive breastand colon carcinoma were selected based upon histologic evaluation. Forthe tumor sections histologic fields of tissue which contained invasivetumor and stroma were selected, but not normal epithelium or significantnumbers of inflammatory cells. The control sections of normal tissuecontained epithelium and a thin section of underlying stroma. Theproportion of epithelial and stromal tissue was similar for both normaland tumor sections.

[0064] In the examples microdissection slides were prepared by coveringstandard histology slides with 200 microliters of warm agarose (1%) andover laying a cover slip. After five minutes the coverslip was removedleaving a thin bed of agarose on the slide. Twenty micron thick frozensections were prepared in a cryostat and placed on the agarose gel. Thetissue was briefly dipped in eosin. Optimum microdissection was achievedby starting at the edge of each section and systematically dissectingand separating histologic fields of interest with the microdissectingdevice of FIG. 3. Areas of interest were retained on the slide forsubsequent analysis. The DNA content of the specimens was determined byspectrophotometric measurement at 260 nm. The DNA content of each samplewas proportional to the number of cells counted in each histologicsection.

EXAMPLE 1

[0065] In this example, samples of normal and tumor tissue matched forcell number were analyzed from each subject. Levels of MMP-2 weredetermined by zymography and quantified using an Arcus scanner. Resultswere statistically analyzed using the students t-test. Cathepsin Blevels were determined as V_(max) against the substrate Z-Arg-Arg-NHMec.

[0066] The results of this example are set forth in Table 1 below whichlists the cathepsin B activity in matched pairs of invasive coloncarcinoma/normal epithelium, and invasive breast carcinoma/normalepithelium. Activity measurement are expressed as V_(max), nmol/min×mgDNA. Cathepsin B activity was increased an average of 2.3 fold in thecolon tumors (p<0.005), and 6.9 fold in the breast tumors (p=0.077)TABLE 1 SAMPLE NORMAL TUMOR TUMOR/NORMAL CATHEPSIN B ACTIVITY ININVASIVE HUMAN COLON CARCINOMA 1 1.38 4.75 3.4 2 1.89 2.25 1.2 3 1.986.32 3.2 4 0.49 1.88 3.8 5 0.44 0.72 1.6 6 1.03 1.92 1.9 7 0.47 1.35 2.98 0.19 0.33 1.7 9 1.07 0.90 0.8 10  0.33 0.88 2.7 Average 0.93 2.13 2.3CATHEPSIN B ACTIVITY IN INVASIVE HUMAN BREAST CARCINOMA 1 0.63 3.02 4.82 0.51 10.08 19.8 3 0.61 4.43 7.3 4 2.21 2.38 1.1 5 2.06 3.72 1.8Average 1.20 4.73 6.9

[0067] As can be seen from Table 1, all five breast tumors and nine ofthe ten colon tumors showed increased activity of cathepsin B ascompared to matched numbers of normal cells from the same patient (Table1). Increased activity in the colon tumors ranged from 19% to 283%, withan average increase in tumors of greater than two fold. The increase ofcathepsin B activity was more pronounced in breast tumors with anaverage increase of slightly less than seven fold.

EXAMPLE 2

[0068] In this example, polymerase chain reaction (PCR) analysis waspreformed. On the basis of previously reported cDNA sequences of 72 kDatype IV collagenase, sense and antisense oligonucleotide primers weresynthesized for amplification of the enzyme activation site (M. Onistoet al, “Reverse Transcription-Polymerase Chain Reaction Phenotyping ofMetalloproteinases and Inhibitors in Tumor Matrix Invasion”, Diagn. Mol.Pathol, 2(2):74-80, 1993). The paired oligonucleotide sequences were:5′-CAA TAC CTG AAC ACC TTC TA, 3′-CTG TAT GTG ATC TGG TTC TTG. LabeledPCR for Single Strand Conformation Polymorphism (SSCP) was obtained bycombining the following in a 10 microliter reaction: 1 microliter 10×PCRbuffer (100 mM Tris-HCL, pH 8.3; 500 mM KCl; 15 M MgCl₂; 0.1% w/vgelatin); 1 microliter of DNA extraction buffer; 50 pmol of each primer;20 nmol each of dCTP, dGTP, dTTT, and dATP; 0.2 microliter [³²P]dCTP(6000 Ci/mmol); and 0.1 unit Taq DNA polymerase. The amplificationreaction was carried out for 30 cycles at 95° C. for 30 s, 60° C. for 30s, and 72° C. for 30 s.

[0069]FIG. 6a shows the expression of MMP-2 in ten invasive coloncarcinoma cases as compared to normal colonic mucosa from the samepatients. The bar graphs show increases of approximately three fold inthe 72 kDa pro-form of the enzyme (p<0.001) and ten fold in the 62 kDaactive form of the enzyme (p<0.001).

[0070]FIG. 6b shows the expression of MMP-2 in five cases of invasivebreast carcinoma. The bar graphs show an appropriate increase of threefold in the 72kDa pro-form of the enzyme (p<0.05) and ten fold in the 62kDa active form of the enzyme (p<0.05).

[0071] The 72 kDa pro-type IV collagenase and 62 kDa active form of theenzyme were increased in all ten colon tumors and all five breast tumorsas compared to normal tissue from the same patient. The increase wasgreater in the 62 kDa active form of the enzyme which was elevated anaverage of ten-fold in both the colon and breast tumors as compared tonormal control tissue. The 72 kDa pro-enzyme levels were increased anaverage of three fold in both tumor types. For both breast and colontumors the increase in the 62 kDa active enzyme was more variable thanthat of the pro-enzyme. Elevations in the 62 kDa active enzyme in tumorsranged from 3 to 20 fold while increases in the 72 kDa pro-enzyme wereconsistently in the 2 to 5 fold range. These results are similar to therecent findings of Davis et al (“Activity of Type IV Collagenases inBenign and Malignant Breast Disease”, Br. J. Cancer, 67:1126-1131, 1993)in their analysis of human breast tumors. These authors performedzymogram analysis of tissue sections from human breast cancer patients.These analyses demonstrated that the fraction of total MMP-2 present asthe 62 kDa activated form was statistically elevated in malignantdisease, and a high proportion of this active enzyme species wasdetected in higher grade tumors. The present invention extends thisanalysis by comparing and quantitating both 72 kDa and 62 kDa forms ofthe enzyme in specific regions of invasive tumor and matched normalcontrol epithelium from the same patient.

EXAMPLE 3

[0072] In this example, strand conformation polymorphism (SSCP) analysiswas preformed. Labeled amplified DNA was mixed with an equal volume offormamide loading dye (95% formamide; 20 mM EDTA; 0.05% bromophenolblue, and 0.05% xylene cyanol). The samples were denatured for 5 min at95° C. and loaded onto a gel consisting of 6% acrylamide (49:1acrylamide:bis), 5% glycerol, and 0.6×TBE. Samples were electrophoresedat 8 W at room temperature overnight. Gels were transferred to 3 mmWhatman paper, dried and autoradiography was performed with Kodak X-OMATfilm.

[0073]FIG. 7 shows SSCP analysis of MMP-2 activation site. The figureshows representative cases of normal colon is mucosa compared toinvasive colon carcinoma, and normal breast tissue compared to invasivebreast carcinoma. No difference is observed between the normal and tumorspecimens. The two band in each lane represent single and double formsof DNA. Similar results were obtained for ten colon carcinomas and fourbreast carcinomas.

[0074] To assess if increased tumor levels of activated MMP-2 are due toa mutation in the enzyme, PCR was used to amplify DNA sequence codingfor the activation site of gelatinase A from the colon and breasttumors. The activation site is located 10 kDa from the N-terminus of theenzyme and contains the site of cleavage which converts the 72 kDapro-enzyme into the 62 kDa active species. Amplification and analysis ofthis region by PCR and SSCP showed no detectable mutations in any of theten colon tumors or four breast tumors studied. These results suggestthat increased levels of active enzyme in invasive tumors is most likelydue to a tumor associated activating species. The sensitivity of PCRamplification of DNA from microdissected frozen tissue sections wasdetermined to be less that one high power field. Similar to theamplification of DNA, amplification of mRNA from small cell populationswas preformed according to the present invention using reverse PCR.

[0075] A previous study indicated that MMP-2 is up-regulated in humancolon carcinoma. However, recently several studies using in situhybridization analysis report that the MRNA level of MMP-2 in humancolon carcinoma is increased in the stromal cells as opposed to thetumor cells. In order to address this possibility frozen tissue sectionswere microdissected to measure enzyme levels of MMP-2 in separate tumorand stromal cell populations. From a single high power field sufficienttissue was recovered to quantitate enzyme levels by zymography. Studiesof invasive tumor cells and adjacent stroma from three cases indicatethat 72 kDa pro-MMP-2 and active 62 kDa form are associated with bothtumor cell and stromal cell populations. Preliminary data suggest thatthe highest enzyme levels are at the tumor-stromal interface.

[0076] According to a preferred embodiment, the present invention isdirected to adhesive transfer methods which involve microscopicvisualization and transfer of cellular material to a procurement ortransfer surface.

[0077] According to the general procedure, an adhesive surface is placedin contact with the surface of the cells or tissue and the adhesiveforce binds the cellular material of interest to the adhesive surface.The adhesive surface which can be the tip of a tool or needle is used toprocure the material and transfer it to a liquid analysis reactionmixture. Examples of adhesive surfaces include adhesive coatings on thetip of the tool, or the use of electrostatic forces between the tip andthe surface of the cellular material.

[0078] As described in detail below, the isolation and transfer methodsof the present invention can involve a specialized continuousactivatable adhesive layer or surface which is applied to the cellularmaterial over an area larger than the area selected for microscopicprocurement. The adhesive function of the subsection of the surface incontact with the area selected for procurement is activated byelectromagnetic or radiation means. According to a preferred embodimenta laser or other electromagnetic radiation source is used to activatethe adhesive forces between the cellular material and the activatableadhesive layer or surface. This allows for accurate generation ofadhesive forces only in the precise microscopic area selected. Suitablelasers include diode-pumped Nd, YAG and Nd:Yag, tunable single frequencyTi:sapphire lasers, solid state lasers. Lasers having wavelength outputsfrom ultraviolet to infrared can be used according to the presentinvention.

[0079] In addition to lasers, it is possible to activate adhesive layerutilizing electrically heated radiation heaters or heated probes,focused or masked non-laser light sources such as flashbulbs, xenonlamps, etc.

[0080]FIGS. 8a-8 d are schematic illustrations of the sequential stepsof an adhesive transfer method according to one embodiment of thepresent invention.

[0081] As depicted in FIG. 8a, the adhesive transfer method utilizes atransfer surface 30 which includes a backing layer 31 and an activatableadhesive layer 32. In procedures which utilize laser activation of theadhesive layer, the backing layer 31 is preferably transparent, e.g.made of a transparent polymer, glass, or similar material. Theactivatable adhesive layer 32 can be an emulsion layer, a coated film,or a separate impregnated web fixed to the backing layer. Examples ofmaterials from which the adhesive layer 32 can be make include thermalsensitive adhesives and waxes (e.g., #HAL-2 180C from PrecisionCoatings), hot glues and sealants (available from Bay Fastening Systems,Brooklyn, N.Y.), ultraviolet sensitive or curing optical adhesives(e.g., N060-N0A81, ThorLabs Inc.), and thermal or optical emulsions(e.g., silkscreen coated emulsion B6 Hi Mesh, Riso Kagaku Corp.)

[0082] The backing layer 31 provides physical support for the adhesivesurface, and thus can be integrated physically into the activatableadhesive surface.

[0083] The activatable adhesive layer 32 is characterized by its abilityto be stimulated (activated) by electromagnetic radiation so as tobecome locally adherent to the tissue. For purposes of selectivelyactivating the activatable adhesive layer 32 one or more chemicalcomponents can incorporated into the layer, which chemical componentscause selective absorbance of electromagnetic energy.

[0084] As depicted in FIG. 8a, the transfer surface 30 is initiallypositioned over a cellular material sample 33 which can be a microtomesection or cell smear which is supported on a support member 34 whichcan be a microscopic slide. In the case of a tissue microtome, routineprocedures can be used to provide paraffin embedded, formalin-fixedtissue samples.

[0085] As shown in FIG. 8b, the transfer surface 30 is brought intocontact with the cellular material sample 33. It is noted that theactivatable adhesive layer 32 preferably has a larger area than thesubregion of cellular material sample which is subsequently selected forprocurement.

[0086] The transfer surface 30 can be fixed to the cellular materialsample support 33 by clips, guides, tape, standard adhesives, or similarconvenient means. The transfer surface 30 can also contain a labelregion 35 (see phantom lines in FIG. 8b) to write information such asthe patient's identification code or a test designation.

[0087] After the transfer surface 30 is brought into contact with thecellular material sample 33, the cellular material sample is viewed bystandard low or high power microscopy to locate the region of interest“A”. This region can range in size to an area smaller than a single cell(less than 10 microns), to a few cells, to a whole field of cells ortissue. When the area of interest “A” is identified, the precise regionof the activatable adhesive layer 32 which is immediately above region“A” is activated by a beam of electromagnetic energy 36, e.g. a laserbeam, sa depicted in FIG. 8c.

[0088] Application of the electromagnetic energy 36 causes the region ofthe activatable adhesive layer 32 which is immediately above region “A”to adhere to region “A”. Although FIGS. 8c and 8 d depict a singleregion of interest “A”, it is to be understood that multiple,discontinuous regions of interest could be selected and procured byappropriate aiming and application of the electromagnetic energy.

[0089] As depicted in FIG. 8d, after one or more regions of interest areidentified and the corresponding region(s) of the activatable adhesivelayer 32 is activated by a beam of electromagnetic energy 36, thetransfer surface 30 is detached from the cellular material samplesupport 34. As shown, the removed transfer surface 30 carries with itonly the precise cellular material from the region of interest “A”,which is pulled away from the remaining cellular material sample.

[0090] As mentioned above, a single transfer surface can be used toremove a plurality of areas of interest from a single cellular materialsample. The transfer surface 30 carrying the procured cellular materialcan be treated with suitable reagents to analyze the constituents of thetransferred material. This can be accomplished by submerging thetransfer surface 30, to which the procured cellular material is adhered,in a suitable reagent solution. Alternatively, one or more of theprocured cellular material regions can be removed from the transfersurface 30, or portions of the transfer surface 30 to which the procuredcellular material are adhered can be punched out of the transfer surface30 and analyzed separately.

[0091] In the following Examples 4 and 5, the following sampleprocurement method was followed. 5-10 micron sections of formalin-fixed,paraffin-embedded tissue or froze tissue were prepared on a glass slideaccording to conventional surgical pathology protocol. The paraffinsections were deparaffinized with xylene (×2), 95% ethanol (×2), 50ethanol (×2), distilled water (×2), and air dried. Frozen or paraffinsections were stained briefly in eosin (1% eosin in 80% ethanol) and airdried.

[0092] An adjacent hematoxylin and eosin section was used to assess thetissue section for optimal areas of microdissection, i.e., localizationof specific small cell populations of interest, exclusion of regionswhich contain significant inflammation, etc.

[0093] Microdissection of selected populations of cells was performedunder direct light microscope visualization. A sterile 30 gage needlewas used as the transfer surface. Electrostatic interaction between theneedle and cellular material provided the adherence needed to removeselected populations of cells. It was determined that pure cellpopulations of as little as 5 cells could be procured. In addition itwas found possible to procure cells arranged as a single cell layer,i.e., normal epithelium, epithelial lining of cystic lesions, etc.

EXAMPLE 4

[0094] Human prostate cancer has been proposed to progress through an insitu tumor phase called prostatic intraepithelial neoplasia (PIN) priorto evolving into overtly invasive cancer. PIN lesions are frequentlyfound in association with prostate carcinoma, and histologically thecells in PION foci have several features similar to those of invasiveprostate cancer cells. Previous reports have shown that PIN lesions arefrequently aneuploid. However the precise relationship between PIN andinvasive carcinoma has remained unclear.

[0095] In this Example, frozen normal and tumor prostate samples from100 patients treated with transurethral prostatectomy or radialprostatomy were collected. Of these, 30 cases which contained clearlyinvasive cancer as well as at least one focus of identifiable PIN wereselected for study during this Example. Fourteen of the set casescontained more than one focus of PIN. The histopathology of the tumorswas variable and included well differentiated, moderately differentiatedand poorly differentiated. PIN lesions were both low and high grade.

[0096] Microdissection of selected populations of normal epithelialcells, cells from PIN lesions, and invasive tumor cells from frozentissue sections was performed under direct light microscopicvisualization utilizing the method discussed above. Specific cells ofinterest were microdissected and procured from unstained 8 μm frozensections. In each case, normal epithelium, PIN cells, and invasive tumorcells from the same patient were analyzed.

[0097] Procured cells were immediately resuspended in a 20 ml solutioncontaining 10 mM Tris-HCL, pH 8.0, 100 mM ethylenediamine tetraaceticacid (EDTA), 1% Tween 20, 0.1 mg/ml proteinase K, and incubatedovernight at 37° C. The mixture was boiled for 5 minutes to inactivatethe proteinase K and 0.5-2% of this solution was used for polymerasechain reaction (PCR) analysis.

[0098] The oligonucleotide primers D8S136, D8S137, and NEFL were used tolocate chromosome 8p12-21. Reactions with D8S137 and NEFL were performedin an MJ Research thermal cycler as follows: 2 minutes at 950° C.,followed by 40 cycles of: 950° C. for 30 seconds, 620° C. for 30seconds, 720° C. for 30 seconds, followed by a final 2 minute incubationat 720° C.

[0099] Reactions with D8S136 were cycled as follows: 2 minutes at 950°C., followed by 40 cycles of: 950° C. for 30 seconds, 550° C. for 30seconds, 720° C. for 30 seconds, followed by a final 2 minute incubationat 720° C.

[0100] PCR was performed in 12.5 ml reactions with 200 mM dNTP, 0.8 mMprimers, 2 μl of alpha [³²P]dCTP (NEN), and 1 unit of Taq polymerase.Labeled amplified DNA was mixed with an equal volume of formamideloading dye (95% formamide; 20 mM EDTA; 0.05% bromophenol blue, and 0.05xylene cyanol).

[0101] The samples were denatured for 5 min at 950° C. and loaded into agel consisting of 7% acrylamide (49:1 acrylamide:bis), 5.7 M urea, 32%formamide, and 0.089 M Tris, 0.089 M borate. 0.002 M EDTA (1×TBE).Samples were electrophoresed at 95 Watts for 2-4 hours. Gels weretransferred to 3 mM Whatman paper, and autoradiography was performedwith Kodak X-OMAT film. The criterion for LOH was complete, or nearcomplete absence of one allele as determined by visualization. Caseswith LOH showed two alleles in the normal epithelium control and oneallele in the tumor or PIN all with similar intensities. Cases withcomplete or near complete loss (i.e., very faint band) of one allele intumor or PIN were considered positive for LOH at that marker.

[0102] The present inventive method was used to microdissect cells fromtissue sections to study loss of heterozygosity on chromosome 8p12-21 inpatients with both prostatic carcinoma and adjacent foci of PIN. Tissuemicrodissection was conducted on 30 patients with concomitant PIN andinvasive prostate cancer. In each case normal epithelium, invasiveprostate cancer and at least one focus of PIN from the same patient wereexamined. In 14 cases multiple foci of PIN were examined. In all caseseach individual PIN lesion and corresponding invasive tumor wereselectively microdissected from of adjacent stroma, normal epitheliumand inflammatory cells. Essentially pure populations of cells ofinterest were procured.

[0103] LOH on chromosome 8p12-21 occurred in at least one PIN lesion in26 of 29 (89.6%) informative cases. Fourteen of the cases contained morethan one PIN lesion. Eleven of these cases showed different allele losspatterns among the PIN lesions, including loss of opposite alleles. Intotal, 8p12-21 LOH was seen in 63.6% (35/55) of PION lesions studied.Allelic loss of chromosome 8p12-21 was seen in invasive tumors in 28 of29 (96.5%) patients. In contrast with the success associated with theadhesive transfer technique of the present invention, the use of ascraping dissection technique produced an LOH of less than 15%. Thisindicates the sensitivity of the adhesive transfer of the presentinvention is much greater than conventional techniques.

EXAMPLE 5

[0104] Nascent in situ breast carcinomas are frequently observed arisingin association with a spectrum of epithelial hyperplasias and invasivecarcinoma. Pathologists have historically interpreted the commonassociation of atypical hyperplasia, in situ carcinoma and invasivecarcinomas as evidence for a relationship among the entities.

[0105] The polymorphic DNA marker used in this Examiner was PYGM locatedon chromosome 11q13. Reactions were cycled in a thermal cycler asfollows: 94° C. for 1.5 min., 55° C. for 1 min., 72° C. for 1 min. for atotal of 35 cycles. PCR was performed in 10 μl volumes and contained 1μl 10×PCR buffer (100 mM Tris-HCl, pH 8.3; 500 mM KCl; 15 mM MgCl₂; 0.1%w/v gelatin; 2 μl of DNA extraction buffer, 50 pM of each primer; 20 nMeach of dCTP, DGTP, dTTP, and dATP; 0.2 μl [³²P ]dCTP (6000 Cl/mM); and0.1 unit Taq DNA polymerase. Labeled amplified DNA was mixed with anequal volume of formamide loading dye (95% formamide; 20 mM EDTA; 0.05%bromophenol blue; and 0.05% xylene cyanol). The samples were denaturedfor 5 min. at 95° C. and loaded into a gel consisting of 6% acrylamide(49:1 acrylamide:bis). Samples were electrophoresed ar 1800 volts for2-4 hours. Gels were transferred to 3 mM Whatman paper, dried andautoradiography was performed with Kodak X-OMAT film. The criterion forLOH from the microdissected in situ and invasive breast samples wascomplete absent of an allele.

[0106] Using the adhesive transfer technique of the present invention,cells were microdissected from normal epithelium, in situ carcinoma andinvasive carcinoma from 8 μm thick formalin fixed deparaffinizedsections from individual biopsies. Allelic loss of chromosome 11q13 wasfound in 69% of human breast carcinoma cases studied (n=105). Theallelic loss was observed in both the in situ and invasive components ofthe tumors. In all cases (26/28) where in situ and invasive cancer waspresent in the same section, the identical allele was lost in the insitu and the invasive carcinoma. This provides molecular support for thelong held hypothesis that in situ breast cancer is a precursor toinvasive cancer.

[0107] In order to finely map the LOH locus on chromosome 11q13, Genomecenter provided a series of SSCP probes mapped to the relevant region ofchromosome 11. The initial LOH area was determined to be bracketed bythe proximal marker PYGM, and by the distal marker INT-2. A subset of 20of the 105 cases exhibited LOH of either INT-2 or PYGM, but not both.Using these special cases, a series of intervening markers were used tomap the smallest overlapping region between INT-2 and PYGM which showsLOH. It has been possible to pinpoint the LOH zone to a regionencompassed by only one or two YAG or Cosmid clones at a location whichoverlaps with the MEN-1 (Multiple Endocrine Neoplasia type 1) locus.

[0108]FIG. 9 shows the results of a sequencing gel electrophoresis ofPCR amplified DNA from human tissue microdissected by the methoddepicted in FIGS. 8a-8 d. Each numbered lane in FIG. 9 is from anindividual dissection. Electrostatic transfer is shown in lanes 17-20.Lanes 18-20 show complete loss (positive LOH) of the top allele comparedto the microdissected control DNA in lane 17. Thermal infrared transfer(conducted as shown in FIGS. 8c and 8 d) is shown in lanes 22-25. Lane22 (control) shows no DNA transfer without activation. Lanes 23 and 24show complete loss (positive LOH) of top allele group compared tocontrol DNA lane 25.

[0109] The above results indicate that microdissection of frozen tissuesections allows for more specific analysis of cell populations withinhuman tumors than by conventional techniques. The microdissectiontechnique of the present invention may be used in combination with anumber of different technologies that allow for analysis of enzymes,MRNA and DNA from pure populations or subpopulations of particular celltypes.

[0110] For example, DNA can be microdissected, using the techniques ofthe present invention, from normal epithelium, pre-malignant lesions,and invasive cancer in the same patient's single tissue sections. THisRNA can then be used to generate differential gene expression librariesby standard RT PCT. These libraries represent cellular stages of cancerprogression. As such, they can be used to screen for cancer diagnosticand prognostic markers.

[0111] This simple technique may have utility in characterizing proteasedistribution during human tumor invasion, precisely determining proteaseexpression in tumor and/or stromal cell populations as an indicator oftumor aggressiveness, and monitoring the effectiveness of anti-proteasetherapeutic agents in inhibiting protease activity at the tumor-stromalinterface. In addition, combination of this microdissection techniquewith PCR, RT PCR, differential display and SSCP may identify geneticalterations in specific subpopulations of tumor or stromal cell thatwould not be evident in heterogeneous human tumor samples.

[0112] The present invention has applications in routine diagnosis ofhuman tumors including microdissection of pre-malignant lesions of alltypes of cancer, genetic analysis of infectious diseases, gene therapy,tissue transformation, and gene localization and analysis of transgenicanimals. Additional applications of this technique include analysis ofthe genotype, cellular products, or infesting organisms of rarepopulations such as Monocytes infected with drug resistant organisms,Reed-Stemberg cells of Hodgkins disease, Kaposi's sarcoma cells, stemcells, and vessel cells. Moreover, genetic analysis, or identificationof, micro-organisms infesting microscopically visualized cells intissues, lymph nodes or inflammatory areas can also be accomplished withhigh precision

[0113] Although the present invention has been described with referenceto particular means, materials and embodiments, from the foregoingdescription, one skilled in the art can easily ascertain the essentialcharacteristics of the present invention and various changes andmodifications may be made to adapt the various uses and characteristicswithout departing from the spirit and scope of the present invention asdescribed by the claims which follow.

1. A method of direct extraction of cellular material from a tissuesample which comprises: providing a tissue sample; contacting saidtissue sample with a transfer surface which can be activated to provideselective regions thereof with adhesive characteristics; identifying atleast one portion of said tissue sample which is to be extracted;activating a region of said transfer surface which is in contact withsaid at least one portion of said tissue sample so that said activatedregion of said transfer surface adheres to said at least one portion ofsaid tissue sample; and separating said transfer surface from saidtissue sample while maintaining adhesion between said activated regionof said transfer surface and said at least one portion of said tissuesample so that said at least one portion of said tissue sample isextracted from a remaining portion of said tissue sample.
 2. A method ofdirect extraction of cellular material from a tissue sample according toclaim 1 , wherein said at least one portion of said tissue samplecomprises a plurality the different portions of said tissue sample.
 3. Amethod of direct extraction of cellular material from a tissue sampleaccording to claim 1 , wherein said adhesive characteristics comprisechemical adhesive properties.
 4. A method of direct extraction ofcellular material from a tissue sample according to claim 1 , whereinsaid adhesive characteristics comprise electrostatic adhesiveproperties.
 5. A method of direct extraction of cellular material from atissue sample according to claim 1 , wherein said activating comprisesapplying electromagnetic energy to said region of said transfer surface.6. A method of direct extraction of cellular material from a tissuesample according to claim 1 , wherein said activating comprises applyingheat to said region of said transfer surface.
 7. A method of directextraction of cellular material from a tissue sample according to claim1 , wherein said identifying comprises visualizing said tissue samplewith a microscope.
 8. A method of direct extraction of cellular materialfrom a tissue sample according to claim 7 , wherein said identifying andextraction is automated.
 9. A method of direct extraction of cellularmaterial from a tissue sample according to claim 1 , further comprisinganalyzing said extracted portion of said tissue sample.
 10. A method ofdirect extraction of cellular material from a tissue sample according toclaim 9 , wherein said extracted portion of said tissue sample and saidactivated region of said transfer surface which is adhered thereto areseparated from a remaining portion of said transfer surface.
 11. Amethod of direct extraction of cellular material from a tissue sampleaccording to claim 1 , wherein said transfer surface comprises a backinglayer and an activatable adhesive layer provided an said backing layer.12. A method of direct extraction of cellular material from a tissuesample according to claim 11 , wherein said backing layer istransparent.
 13. A method of direct extraction of cellular material froma tissue sample according to claim 11 , wherein said backing layerinclude a label area.
 14. A method of direct extraction of cellularmaterial from a tissue sample according to claim 1 , wherein said tissuesample comprises a microtome section.
 15. A method of direct extractionof cellular material from a tissue sample according to claim 1 , whereinsaid tissue sample comprises a cellular smear.
 18. A method of directextraction of cellular material from a tissue sample according to claim1 , wherein said activating comprises irradiating said region of saidtransfer surface with laser-derived energy. (Ser. No. 08/544,388)